Mechanistic and Structural Analysis of <i>Drosophila melanogaster</i> Arylalkylamine <i>N</i>‑Acetyltransferases

Abstract

Arylalkylamine <i>N</i>-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other <i>N</i>-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in <i>Drosophila melanogaster</i> for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in <i>D. melanogaster</i>, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both <i>D. melanogaster</i> AANAT variants (AANATA and AANATB) in <i>Escherichia coli</i> and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH–activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent p<i>K</i>_a of 7.0. Using the data generated for the kinetic mechanism, structure–function relationships, pH–rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA